Occurrence of Acanthamoeba Genotypes in Wastewater Samples in Tehran, Iran.

Background
Acanthamoeba spp. is potentially pathogenic free-living amoeba that can exist in various water sources. The presence of this amoeba in water sources could be a health hazard as Acanthamoeba could lead to severe diseases such as Acanthamoeba keratitis and encephalitis. This study aimed to determine the genotypes of isolated Acanthamoeba spp. in raw wastewater samples in Tehran, Iran.


Methods
Overall, 90 raw wastewater samples were collected from water treatment facilities in west and south of Tehran, Iran during 2014-2016. Water samples were filtered and cultured on non-nutrient agar (NNA) medium enriched with Escherichia coli. Morphological and molecular analyses were done on positive strains. The pathogenic ability of the isolated strains was determined using physical assays.


Results
Totally, 6 out of 90 (6.7%) samples were positive for Acanthamoeba, according to morphological characteristics of double-walled cysts. Genotyping and sequencing of the positive strains showed Acanthamoeba belonging to T4 (83%) and T11 (17%) genotypes. In vitro pathogenicity tests were revealed that five isolates were classified as non-pathogenic strains and one strain belonging to T4 genotype was classified as the highly pathogenic amoebae.


Conclusion
The current research reflected a low contamination of wastewater sources to Acanthamoeba. More studies regarding the contamination of wastewaters before and after treatment are required in different places of the country.


Introduction
canthamoeba spp. are one of the most prevalent protozoa with worldwide distribution, these amphizoics amoebae were isolated from various environmental sources such as different kinds of waters including seawater, tap water, aquarium, fresh and saltwater lakes, hot spring resorts, bottled water and swimming pool (1,2). To date, the classification of Acanthamoeba using molecular approaches revealed 20 genotypes (T1-T20) based on 18S rDNA gene region (3), most genotypes have been isolated from clinical cases, however, T4 genotype is the most common strain among the clinical and environmental specimens. The T4 genotype has greater pathogenic ability among the others (1,4). Moreover, T4 genotype is the most common genotype isolated from corneal scrapes of keratitis patients in Iran (5,6).
Acanthamoeba spp. acting as opportunistic amoebeae is able to develop two severe diseases including Granulomatous Amoebic Encephalitis (GAE) and sight-threatening corneal infection termed Acanthamoeba keratitis (AK) (1). The major risk factor for AK is the usage of contact lenses and exposure to contaminated water. Moreover, swimming or bathing in the contaminated aquatic environment is hazardous to contact lens wearers (1,2). Since Acanthamoeba could act as a reservoir for pathogenic microorganism, this could double the threat for human health (1,7). Several studies in different parts of Iran showed the occurrence of Acanthamoeba genotypes and other free-living amoebae (FLA) in the various sources of waters. The presence of Acanthamoeba was revealed in recreational water and tap water sources in northern and southern Iran, Gilan and Kish Island, respectively. The results showed the occurrence of T4 genotype in recreational waters and T3, T4, T5 and T11 genotypes in tap water sources (8,9). Studies on river waters and hot springs in the country were also detected T3, T4, T5 and T15 genotypes of Acanthamoeba and Naegleria (10)(11)(12)(13).
Despite numerous studies conducted on water sources in Iran, there were no data regarding the contamination of wastewater sources to Acanthamoeba spp. in the region and thus the main aim of the current study was to determine the occurrence of Acanthamoeba genotypes in wastewater of two treatment facilities in west and south of Tehran Province. The pathogenic potential of the isolated strains were also evaluated using thermos and osmotolerance assays.
This study aimed to determine the genotypes of isolated Acanthamoeba spp. in raw wastewater samples in Tehran, Iran.

Sampling
During 2014-2016, 90 raw wastewater samples were collected from water treatment facilities in west and south of Tehran Province. Fifty water samples were collected from the west of the Tehran, Shahrak-e Ekbātān, and 40 samples were obtained from the south of Tehran, Chahardonge region, both areas are located in the urban region. All of the specimens were transferred to Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Iran.

Sample processing and microscopic survey
Filtration of each sample was performed using nitrocellulose membranes (45-µm diameter). The filters were cultured on 1.5% nonnutrient agar (NNA) medium plates with heatinactivated Escherichia coli (14). The plates monitoring was performed daily using an inverted microscope (15). Cloning of the posi-A tive plates was done to purify the amoebae from bacterial and fungal contamination (5).

In vitro pathogenicity tests
Osmo-tolerance and thermo-tolerance assays were performed on the positive strains. For osmotolerance test, non-nutrient agar with 1 M and 0.5 M mannitol concentration was used to evaluate the outgrowth of amoebae (16). For thermo-tolerance assay, the growth of amoebae was surveyed by the inverted microscopy. The temperature was set at 37 and 42 °C for a week (17). All the plates were monitored for one week.

DNA extraction, PCR amplification, and Sequencing
Acanthamoeba spp. were harvested using sterile phosphate buffered saline (PBS), at pH 7.2, then the harvested amoebae were concentrated and lysed using lysozyme and glass beads treatment. High Pure polymerase chain reaction (PCR) Template Preparation Kit (Roche, Mannheim, Germany) was used for DNA extraction. PCR amplified the ASA.S1 region (500 bp) of 18S rRNA gene, along with primers JDP1 5′-GGCCCAGATCGTTTACCGTGAA-3′ and JDP2 5′-TCTCACAAGCTGCTAGGGAGTCA-3′ (16). A total of 30-μl volume of PCR reaction was fixed by using Amplicone (Taq DNA Polymerase Master Mix RED, Denmark), 0.1 μM of each primer, distilled water, and DNA template. The cycling conditions stared with 94 °C for 1 min; 35 cycles of 94 °C for 35 sec, annealing step was 56 °C for 45 sec and 72 °C for 1 min; final extension was 72 °C for 10 min. The presence of PCR product bands was confirmed with UV light after electrophoresis and staining of PCR product. Purification and sequencing were done using the ABI 3130X sequencer. The sequences adjusted with chromas (ver. 1.0.0.1), afterward compared with BLAST GenBank database.
In vitro pathogenicity tests including osmotolerance and thermo-tolerance assays were confirmed that only a single isolate (TW1) was highly pathogenic as the strain showed a good growth at high temperature and osmolarity, while the other strains were classified as non-pathogenic amoebae ( Table  1).

Discussion
This study is the first survey of Acanthamoeba spp. isolated from the wastewater specimens in the country. Our study showed 6.7% of the samples were positive for Acanthamoeba spp., in which 83% belonged to T4 genotype. The data of this study reflected the occurrence of Acanthamoeba spp. in the wastewater of the capital in the country. However, despite the other environmental studies in Iran, the current research reflected a low contamination of wastewater sources to Acanthamoeba. This may be due to inhibitory effect of the contaminant in the polluted water. Only Acanthamoeba was detected in the studied samples and other freeliving amoebae were not observed. This can be explained by more resistance of Acanthamoeba cysts in harsh conditions and the fact that other free-living amoebae are more fragile (18). The result of the other studies is opposite to ours. A study conducted in wastewater treatment plant (WWTP) process and sludge in agriculture in the Navarra Community of Spain region showed Acanthamoeba spp. as the most common protozoa in outlet water and sludge (19). Moreover, another research in wastewater treatment plant of central Spain detected Acanthamoeba belonged to T16 and T7 genotypes. Moreover, phylogenetic analysis detected T19 genotype among the samples (20). Another research in South Africa showed the occurrence of free-living amoebae (87.2%) in wastewater treatment plant (21). The current study showed the presence of T4 and T11 genotypes in the contaminated water samples.
Acanthamoeba belonging to T3, T4, T7 and T9 genotypes, Hartmannella and Naegleria were isolated from the Spanish wastewater treatment plants (22). Moreover, the most frequent FLA was the genus Acanthamoeba (59%) from wastewater in the north of Mexico City (23). Despite the mentioned researchers in the world, there were no data regarding Acanthamoeba spp. distribution of wastewater samples in Iran.
Most of the wastewaters recovered after treatment and reused directly for irrigation systems, river flow augmentation and industrial consumption (13,24). As expected, the present study reflected that the most common strain belonged to T4 genotype. T4 genotype was also the frequently isolated strain from clinical and environmental samples (5,6,10). However, the interesting point in the present research was the low pathogenicity of most strains. This is in agreement with study that revealed not all T4s have pathogenic potential (25). Moreover, nine strains from nasal swabs of immunocompromised patients belonged to T4 genotype were classified as non-pathogenic ones (16).
Acanthamoeba belonged to T11 genotype (A. hatchetii) was another isolated strain in the wastewater specimens, this genotype was reported from the keratitis patients in Iran and isolated from the environmental samples previously (6,26). However, this strain showed low pathogenic potential. More studies regarding the pathogenic ability of the isolated strains such as in vivo tests and animal-based studies are of importance.

Conclusion
The data of current study was reflected the low occurrence of Acanthamoeba genotypes in the raw wastewater samples. More studies about the contamination of wastewaters before and after treatment are required in different places of the country.